Muscle invasive bladder cancer (MIBC) affects approximately 20,000 patients each year in the United States alone, and is associated with significant morbidity and mortality. Yet, despite its significance, it has been relatively neglecte in basic cancer research, and as a consequence, many salient issues regarding its molecular and cellular origins have not been fully elucidated. In particular, the cell(s) of origin of bladde cancer and consequences of specific oncogene/tumor suppressor alterations in these cells of origin have not been systematically investigated. To a large extent, this reflects the limitations f current model systems for pursuing such studies. In the current proposal, we describe innovative genetically-engineered mouse models for investigating the cell type(s) of origin of bladder cancer. In particular, we have established an approach to achieve inducible gene targeting in specific cell types in the bladder urothelium, which will enable our studies of the cel of origin in vivo as well as their isolation for tumor engraftment assays and explant approaches. In addition, we have established novel culture methods to derive patient-derived organoids to model human bladder cancer in explant culture, which will provide a vital resource for extrapolating our findings from mouse to man using integrative systems biology approaches. Thus, we are ideally poised to investigate the hypothesis that the cell type of origin for bladder cancer, combined with specific oncogene/tumor suppressor alterations, can generate tumor subtypes that are associated with distinct treatment responses and/or patient outcomes. Toward this end, we will pursue the following Specific Aims: In Aim 1, we will systematically analyze the tumorigenic potential of urothelial cell types in GEM models in vivo, and will perform cross-species systems analyses of these findings compared with patient-derived human organoids. In Aim 2, we will investigate whether tumorigenic properties of potential cell type(s) of origin are defined by specific oncogene/tumor suppressor alterations. In particular, we will evaluate the oncogenic activities of ERBB2 (HER2/Neu) and the tumor suppressor activity of TSC1, both of which are altered in MIBC, in order to investigate their functional relevance in orthotopic models and ultimately in GEM models. In Aim 3, we will perform preclinical studies to determine whether distinct cell types of origin help define treatment response, by focusing on chemotherapy agents that are widely used in the clinic, as well as on agents that target PI3 kinase, which is highly relevant for bladder cancer. Thus, the studies outlined in this proposal will provide a comprehensive analysis of the cell types of origin of bladder cancer, their roles in defining bladder cancer subtypes, and their relevance for treatment response.